DISPERSION, ABERRATION AND DECONVOLUTION IN MULTIWAVELENGTH FLUORESCENCE IMAGES

The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spr ead function as wavelength is varied over the range commonly used in f luorescence microscopy. For a given sample, optical conditions can gen erally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated po int spread functions. Therefore, when multiple fluorophores are used t o localize different components in the same sample, the image of the d istribution of at least one of the fluorophores will be spherically ab errated. This aberration causes a loss of intensity and resolution, th ereby complicating the localization and analysis of multiple component s in a multi-wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative de convolution, as long as the spherical aberration in the point spread f unction used for deconvolution matches the aberration in the image rea sonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolutio n of many biological images can be achieved by collecting a small libr ary of spherically aberrated and unaberrated point spread functions, a nd then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple c omponents can then be accurately studied in a multi-wavelength image.